Tuberculosis is a devastating global health crisis caused by Mycobacterium tuberculosis (Mtb) that claims over 2 million lives each year. This pathogen is able to survive inside macrophages and persist within patients for years, causing latent TB infections (LTBI). Mtb is refractory to antibiotic treatment because latent (or dormant) TB exhibit phenotypic drug resistance due to metabolic and structural adaptations to conditions within pulmonary granuloma lesions. As a result, successful treatment of TB requires a regimen including a cocktail of multiple drugs administered for 6-9 months. The emergence of multi-drug resistant Mtb strains has further complicated the already difficult task of treating TB. Thus, ther is a dire need for potent drugs with novel modes of action capable of shortening the course of treatment and killing drug-resistant and latent Mtb. This proposal seeks to address this critical lack of drugs that effectively kill latent Mtb. In collaboration with Harbor Branch Oceanographic Institute (HBOI), we will exploit the enormous chemical diversity present among secondary metabolites of marine organisms by screening a peak library of marine natural products (MNP) against Mtb in two models of latency. In the R21 phase, we will build upon preliminary studies by screening ~5000 MNP for activity against Mtb in log-phase broth cultures, Mtb growing in macrophages, and dormant Mtb using an in vitro multi-stress model (MSM) of latency (Aim 1). In addition, we will purify and structurally characterize hit fractions bactericidal for Mtb whic we identified in a completed pilot screen (Aim 2). In the R33 phase, we will purify and define the structures of prioritized active compounds from hit fractions from all three screens (Aim 3), Finally, we will conduct detailed characterization of purified lead compounds to determine their potency, specificity, and potential targets and mode of action (Aim 4). We hypothesize that these models will favor the identification of drugs active against latent Mtb that target pathways conditionally essential for survival in vivo. We anticipate that the chemical diversity present in MNP will facilitate the identification of compounds with unique structures, targets, and mechanisms of action. Our long-term goal is the discovery of novel lead compounds that would significantly improve the treatment of latent and drug-resistant tuberculosis.